Identification and characterization of a BRAF fusion oncoprotein with retained autoinhibitory domains

Weinberg, Florian; Griffin, Ricarda; Fröhlich, Martina; Heining, Christoph; Braun, Sandra; Spohr, Corinna; Iconomou, Mary; Hollek, Viola; Röring, Michael; Horak, Peter; Kreutzfeldt, Simon; Warsow, Gregor; Hutter, Barbara; Uhrig, Sebastian; Neumann, Olaf; Reuß, David; Heiland, Dieter Henrik; Kalle, Christof von; Weichert, Wilko; Stenzinger, Albrecht; Brors, Benedikt; Glimm, Hanno; Fröhling, Stefan; Brummer, Tilman

doi:10.1038/s41388-019-1021-1

Cited by 22 publications

(26 citation statements)

References 80 publications

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“…PLX8394 has also shown some efficacy against BRAF protein fusions [106]. BRAF fusions contain the catalytically active CR3 kinase domain but lack the regulatory CR1 and CR2 domains [107]. In some melanoma cell lines driven by BRAF fusions with a 5 0 partner that contains a dimerisation domain, PLX8394 has been shown to paradoxically activate RAF signalling [108].…”

Section: Breaking the Paradoxmentioning

confidence: 99%

Inhibition of RAF dimers: it takes two to tango

Cook

2020

Biochemical Society Transactions

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The RAS-regulated RAF–MEK1/2–ERK1/2 pathway promotes cell proliferation and survival and RAS and BRAF proteins are commonly mutated in cancer. This has fuelled the development of small molecule kinase inhibitors including ATP-competitive RAF inhibitors. Type I and type I½ ATP-competitive RAF inhibitors are effective in BRAFV600E/K-mutant cancer cells. However, in RAS-mutant cells these compounds instead promote RAS-dependent dimerisation and paradoxical activation of wild-type RAF proteins. RAF dimerisation is mediated by two key regions within each RAF protein; the RKTR motif of the αC-helix and the NtA-region of the dimer partner. Dimer formation requires the adoption of a closed, active kinase conformation which can be induced by RAS-dependent activation of RAF or by the binding of type I and I½ RAF inhibitors. Binding of type I or I½ RAF inhibitors to one dimer partner reduces the binding affinity of the other, thereby leaving a single dimer partner uninhibited and able to activate MEK. To overcome this paradox two classes of drug are currently under development; type II pan-RAF inhibitors that induce RAF dimer formation but bind both dimer partners thus allowing effective inhibition of both wild-type RAF dimer partners and monomeric active class I mutant RAF, and the recently developed “paradox breakers” which interrupt BRAF dimerisation through disruption of the αC-helix. Here we review the regulation of RAF proteins, including RAF dimers, and the progress towards effective targeting of the wild-type RAF proteins

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Section: Breaking the Paradoxmentioning

confidence: 99%

Inhibition of RAF dimers: it takes two to tango

Cook

2020

Biochemical Society Transactions

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“…Interestingly, the paradox breakers PLX7904 and PLX8394 were less effective in BRAF fusion containing cells compared to their BRAF V600E positive counterparts. Similarly, Weinberg et al [30] observed that the paradox breakers PLX7904 and PLX8394 were more effective in suppressing MEK/ERK phosphorylation triggered by BRAF V600E than by the TTYH3:BRAF fusion protein. This might be explained by the fact that PLX7904 and PLX8394 were developed with vemurafenib as starting point [55].…”

Section: Discussionmentioning

confidence: 89%

“…Both primers introduce flanking NotI sites into the amplicon, which was subcloned into pSC-A (Stratagene) for further propagation. The cDNA was then recovered by NotI digestion and subcloned into NotI linearized pMIBerry-NotI unique [30] to yield pMIBerry NotI unique/BRAFWT-HA. This retroviral vector allows for the expression of a bicistronic transcript encoding the protein-ofinterest and dsRed2.…”

Section: Plasmidsmentioning

confidence: 99%

A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells

Usta

Sigaud

Buhl

et al. 2020

Molecular Cancer Therapeutics

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Pilocytic astrocytomas (PAs) as well as other pediatric low-grade gliomas (pLGGs) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these singlepathway diseases. We aimed to develop an assay suitable for pre-clinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway suppressing synergistic drug combinations.A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1-binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAF V600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screen of a MAPKi library was performed and synergy of selected combinations was calculated.Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAF V600E mutation backgrounds.We here report a novel reporter assay for medium-to high-throughput pre-clinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAF V600E mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

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“…These genes included six genes (TAGLN2, HSPA5, FN1, TTYH3, GRN and MTPN). Of these genes, TAGLN2 ( Beyer et al., 2018 ; Han et al., 2017 ), FN1 ( Yu et al., 2020 ; Gu, Gu & Shou, 2014 ; Guo, Heller & Thorslund, 2016 ; Liao et al., 2018 ), TTYH3 ( Weinberg et al., 2020 ), GRN, ( Ness, Riemenschneider & Baches., 2009 ; Trigos et al., 2019 ) have been reported in GBM.…”

Section: Resultsmentioning

confidence: 99%

Identification of robust diagnostic and prognostic gene signatures in different grades of gliomas: a retrospective study

Liu

Zhang

et al. 2021

PeerJ

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Background Gliomas are the most common primary tumors of the central nervous system. The complexity and heterogeneity of the tumor makes it difficult to obtain good biomarkers for drug development. In this study, through The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA), we analyze the common diagnostic and prognostic moleculer markers in Caucasian and Asian populations, which can be used as drug targets in the future. Methods The RNA-seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) were analyzed to identify signatures. Based on the signatures, the prognosis index (PI) of every patient was constructed to predict the prognostic risk. Also, gene ontology (GO) functional enrichment analysis and KEGG analysis were conducted to investigate the biological functions of these mRNAs. Glioma patients’ data in the CGGA database were introduced to validate the effectiveness of the signatures among Chinese populations. Excluding the previously reported prognostic markers of gliomas from this study, the expression of HSPA5 and MTPN were examined by qRT-PCR and immunohistochemical assay. Results In total, 20 mRNAs were finally selected to build PI for patients from TCGA, including 16 high-risk genes and four low-risk genes. For Chinese patients, the log-rank test p values of PI were both less than 0.0001 in two independent datasets. And the AUCs were 0.831 and 0.907 for 3 years of two datasets, respectively. Moreover, among these 20 mRNAs, 10 and 15 mRNAs also had a significant predictive effect via univariate COX analysis in CGGA_693 and CGGA_325, respectively. qRT-PCR and Immunohistochemistry assay indicated that HSPA5 and MTPN over-expressed in Glioma samples compared to normal samples. Conclusion The 20-gene signature can forecast the risk of Glioma in TCGA effectively, moreover it can also predict the risks of Chinese patients through validation in the CGGA database. HSPA5 and MTPN are possible biomarkers of gliomas suitable for all populations to improve the prognosis of these patients.

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Identification and characterization of a BRAF fusion oncoprotein with retained autoinhibitory domains

Cited by 22 publications

References 80 publications

Inhibition of RAF dimers: it takes two to tango

Inhibition of RAF dimers: it takes two to tango

A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells

Identification of robust diagnostic and prognostic gene signatures in different grades of gliomas: a retrospective study

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